Cladosporium cladosporioides PCR Detection Kit Product # 33000

Transcription

1 3430 Schmn Parkway Thrld, ON, Canada L2V 4Y6 Phne: (905) Fax: (905) Cladsprium cladspriides PCR Detectin Kit Prduct # Prduct Insert Pathgen Infrmatin Cladsprium is ne f the mst widespread mlds. It includes abut 40 species naturally fund in sil, n decaying plant material and as plant pathgens. Cladsprium rt (Cladsprium spp.) f grapevine (Vitis vinifera) is a cmmn disease, particularly in Cabernet Sauvignn and ther red wine grape cultivars. It is favred by delayed harvest t btain the phenlic maturity necessary fr highquality red wine. Symptms appear n mature grapes and are characterized by berry dehydratin, a firm decay affecting a small prtin f the berry and a superficial live-green mld. Rapid and accurate detectin f Cladsprium infectins is highly imprtant t facilitate the mnitring f Cladsprium in plant samples. Principle f the Test Nrgen s Cladsprium cladspriides PCR Detectin Kit cnstituents a ready-t-use system fr the islatin and detectin f C.cladspriides using end-pint PCR. The kit first allws fr the islatin f fungal DNA frm the plant samples using spin-clumn chrmatgraphy based n Nrgen s prprietary resin. Fungal DNA can be islated frm fungi grwing n culture plates, r frm plant tissue r fruit using this kit. The DNA is islated free frm inhibitrs, and can then be used as the template in a PCR reactin fr C.cladspriides detectin using the prvided C.cladspriides Master Mix. The C.cladspriides Master Mix cntains reagents and enzymes fr the specific amplificatin f a 320 bp regin f the fungal genme. In additin, Nrgen s C.cladspriides PCR Detectin Kit cntains a secnd heterlgus amplificatin system t identify pssible PCR inhibitin and/r inadequate islatin. The amplificatin and detectin f either the Islatin Cntrl (IsC) r the PCR cntrl (PCRC) des nt reduce the detectin limit f the analytical C.cladspriides PCR. This kit is designed t allw fr the testing f 24 samples. Kit Cmpnents: Cmpnent Cntents Lysis Slutin 15 ml Wash Slutin 9 ml Elutin Buffer 3 ml Bead Tubes 24 Mini Filter Spin Clumns 24 Cllectin Tubes 24 Elutin tubes (1.7 ml) 24 C.cladspriides 2x PCR Master Mix 0.35 ml C.cladspriides Islatin Cntrl (IsC)* a 0.3 ml C.cladspriides Psitiv Cntrl (PsC)* b 0.1 ml Nuclease Free-Water 1.25 ml Nrgen s DNA Marker 0.1 ml Prduct Insert 1 * IsC = Islatin Cntrl ; PsC= Psitive Cntrl a The islatin cntrl is a clned PCR prduct. b The psitive cntrl is C.cladspriides genmic DNA

2 Custmer-Supplied Reagents and Equipment Dispsable pwder-free glves Benchtp micrcentrifuge 1.5 ml micrcentrifuge tubes 65 C water bath r heating blck % ethanl 70% ethanl RNase A (ptinal) Lyticase (ptinal) Strage Cnditins and Prduct Stability All buffers shuld be kept tightly sealed and stred at rm temperature (15-25 C). Buffers can be stred fr up t 1 year withut shwing any reductin in perfrmance. The C.cladspriides 2x PCR Master Mix, C.cladspriides Psitive Cntrl (PsC) and the C.cladspriides Islatin Cntrl (IsC) shuld be kept tightly sealed and stred at -20 C fr up t 1 year withut shwing any reductin in perfrmance. Repeated thawing and freezing (> 2 x) shuld be avided, as this may reduce the sensitivity. If the reagents are t be used nly intermittently, they shuld be frzen in aliquts. General Precautins The user shuld exercise the fllwing precautins when using the kit: Use sterile pipette tips with filters. Stre and extract psitive material (specimens, cntrls and amplicns) separately frm all ther reagents and add it t the reactin mix in a spatially separated facility. Thaw all cmpnents thrughly at rm temperature befre starting an assay. When thawed, mix the cmpnents and centrifuge briefly. Wrk quickly n ice. Quality Cntrl In accrdance with Nrgen s ISO 9001 and ISO certified Quality Management System, each lt f Nrgen s C.cladspriides PCR Detectin Kit, including the C.cladspriides 2x PCR Master Mix, C.cladspriides Islatin Cntrl (IsC) and C.cladspriides Psitive Cntrl (PsC) are tested against predetermined specificatins t ensure cnsistent prduct quality. Prduct Use Limitatins Nrgen s C.cladspriides PCR Detectin Kit is designed fr research purpses nly. It is nt intended fr human r diagnstic use. Prduct Warranty and Satisfactin Guarantee NORGEN BIOTEK CORPORATION guarantees the perfrmance f all prducts in the manner described in ur prduct manual. The custmer must determine the suitability f the prduct fr its particular use. Safety Infrmatin Ensure that a suitable lab cat, dispsable glves and prtective gggles are wrn when wrking with chemicals. Fr mre infrmatin, please cnsult the apprpriate Material Safety Data Sheets (MSDSs). These are available as cnvenient PDF files nline at CAUTION: DO NOT add bleach r acidic slutins directly t the sample-preparatin waste.

3 Prtcl A. Cladsprium cladspriides genmic DNA Islatin Imprtant Ntes Prir t Beginning Prtcl: A variable speed centrifuge shuld be used fr maximum kit perfrmance. If a variable speed centrifuge is nt available a fixed speed centrifuge can be used, hwever reduced yields may be bserved. Ensure that all slutins are at rm temperature prir t use, and that n precipitates have frmed. If necessary, warm the slutins and mix well until the slutins becme clear again. Prepare a wrking cncentratin f the Wash Slutin by adding 21 ml f % ethanl (prvided by the user) t the supplied bttle cntaining the cncentrated Wash Slutin. This will give a final vlume f 30 ml. The label n the bttle has a bx that may be checked t indicate that the ethanl has been added. Lysate can be prepared frm either fungi grwing n plates, plant tissue r fruit. Please ensure that yu fllw the prper prcedure fr lysate preparatin in Step 1a. Fr the islatin f genmic DNA frm fungi grwing n plates, Cllectin Slutin must be prepared. Cllectin Slutin cnsists f 0.9% (w/v) NaCl prepared with distilled water. Preheat a water bath r heating blck t 65 C. C. cladspriides Islatin Cntrl (CC IsC) A Cladsprium cladspriides Islatin Cntrl (IsC) is supplied. This allws the user t cntrl the DNA islatin prcedure. Fr this assay, add the C. cladspriides Islatin Cntrl (IsC) t the lysate during the islatin prcedure The C. cladspriides Islatin Cntrl (IsC) must nt be added t the sample material directly. D nt freeze and thaw the C. cladspriides Islatin Cntrl (IsC) mre than 2 times. The C. C. cladspriides Islatin Cntrl (IsC) must be kept n ice at all times during the islatin prcedure. The PCR cmpnents f the C. cladspriides PCR Detectin Kit shuld remain at -20 C until DNA is extracted and ready fr PCR amplificatin. 1. Lysate Preparatin a. Fungi Grwing n Plates: Add apprximately 5 ml (vlume can be adjusted based n density f fungal grwth) f Cllectin Slutin (see ntes befre use) t the plate and gently cllect fungal spres and mycelium with an inculatin lp r autclaved pipette tip, ensuring nt t cllect any agar debris. Transfer up t 1 ml f washed spres and wet mycelium t a micrcentrifuge tube (prvided by user). Fungi frm Plant Tissue r Fruit: Wash the tissue r fruit with an apprpriate amunt f DNAse free water with vrtexing. Transfer up t 1 ml f washed spres and wet mycelium t a micrcentrifuge tube (prvided by user). b. Centrifuge at 14,000 x g (~14,000 RPM) fr 2 minutes t pellet the cells. Pur ff the supernatant carefully s as nt t disturb r disldge the cell pellet. c. Add 500 µl f Lysis Slutin t the cell pellet. Resuspend the cells by gentle vrtexing. d. Transfer the mixture t a prvided Bead Tube and secure the tube hrizntally n a flat-bed vrtex pad with tape, r in any cmmercially available bead beater equipment (e.g. Scientific Industries Disruptr Genie TM ). e. Vrtex fr 5 minutes at maximum speed r ptimize the cnditin fr any cmmercially available bead beater equipment. Nte: Faming during the hmgenizatin is cmmn. This faming is due t detergents present in the Lysis Buffer and will nt affect the prtcl.

4 f. Incubate the Bead Tube with lysate at 65 C fr 10 minutes. Occasinally mix the lysate 2 r 3 times during incubatin by inverting the tube. g. Briefly spin the tube t remve liquid frm the cap, and transfer all f the lysate, including cell debris, t a DNase-free micrcentrifuge tube (prvided by the user) by pipetting. Ensure that the beads are nt transferred during the pipetting. h. Centrifuge the tube fr 2 minutes at g (~14,000 RPM). i. Carefully transfer clean supernatant t a new DNase-free micrcentrifuge tube (prvided by the user) withut disturbing the pellet. Nte the vlume. j. Add an equal vlume f 70% ethanl (prvided by the user) t the lysate cllected abve (100 µl f ethanl is added t every 100 µl f lysate). Vrtex t mix. k. Prceed t Step 2: Binding t Clumn 2. Binding DNA t Clumn a. Assemble a spin clumn with ne f the prvided cllectin tubes. b. Add 10 µl f C.cladspriides Islatin Cntrl (IsC) t the lysate mixture. c. Apply up t 600 µl f the lysate with ethanl nt the clumn and centrifuge fr 1 minute at 14,000 x g (~14,000 RPM). Discard the flwthrugh and reassemble the spin clumn with the cllectin tube. Nte: Ensure the entire lysate vlume has passed thrugh int the cllectin tube by inspecting the clumn. If the entire lysate vlume has nt passed, spin fr an additinal minute. d. Depending n yur lysate vlume, repeat step 2c if necessary. 3. Clumn Wash a. Apply 500 µl f Wash Slutin t the clumn and centrifuge fr 1 minute. Nte: Ensure the entire wash slutin has passed thrugh int the cllectin tube by inspecting the clumn. If the entire wash vlume has nt passed, spin fr an additinal minute. b. Discard the flwthrugh and reassemble the clumn with its cllectin tube. c. Repeat step 3a t wash clumn a secnd time. d. Discard the flwthrugh and reassemble the spin clumn with its cllectin tube. e. Spin the clumn fr 2 minutes in rder t thrughly dry the resin. Discard the cllectin tube. 4. DNA Elutin a. Place the clumn int a fresh 1.7 ml Elutin tube prvided with the kit. b. Add 75 µl f Elutin Buffer t the clumn. c. Centrifuge fr 2 minutes at 200 x g (~2,000 RPM), fllwed by a 1 minute spin at 14,000 x g (~14,000 RPM). Nte the vlume eluted frm the clumn. If the entire vlume has nt been eluted, spin the clumn at 14,000 x g (~14,000 RPM) fr 1 additinal minute. 5. Strage f DNA The purified DNA may be stred at 20 C fr a few days. It is recmmended that samples be placed at 70 C fr lng term strage.

5 B. Cladsprium cladspriides PCR Assay Preparatin Ntes: Befre use, suitable amunts f all PCR cmpnents shuld be cmpletely thawed at rm temperature, vrtexed and centrifuged briefly. The amunt f C.cladspriides 2X PCR Master Mix prvided is enugh fr up t 32 PCR reactins (24 sample PCR, 4 psitive cntrl PCR and 4 n template cntrl PCR). Fr every PCR run, ne reactin cntaining C. cladspriides Psitive Cntrl (PsC) and ne reactin as n template cntrl must be included fr prper interpretatin f results. The recmmended minimum number f DNA samples tested per PCR run is 6. Using a lwer vlume frm the sample than recmmended may affect the sensitivity f C. cladspriides Limit f Detectin. 1. Prepare the PCR fr sample detectin as shwn in Table 1 belw. The recmmended amunt f sample DNA t be used is 5 µl. Hwever, a vlume between 1 and 10 µl f sample DNA may be used as template. Adjust the final vlume f the PCR reactin t 20 µl using the Nuclease-Free Water prvided. Table 1. PCR Assay Preparatin PCR Cmpnents Vlume Per PCR Reactin C. cladspriides 2X PCR Master Mix 10 µl Sample DNA 2 t 5 µl Nuclease-Free Water Up t 10 µl Ttal Vlume 20 µl 2. Fr every PCR run, prepare ne psitive cntrl PCR as shwn in Table 2 belw: Table 2. PCR Psitive Cntrl Preparatin PCR Cmpnents Vlume Per PCR Reactin C. cladspriides 2X PCR Master Mix 10 µl C. cladspriides Psitive Cntrl (PsC) 10 µl Ttal Vlume 20 µl 3. Fr every PCR run, prepare ne n template cntrl PCR as shwn in Table 3 belw: Table 3. PCR Negative Cntrl Preparatin PCR Cmpnents Vlume Per PCR Reactin C. cladspriides 2X PCR Master Mix 10 µl Nuclease-Free Water 10 µl Ttal Vlume 20 µl

6 C. Cladsprium cladspriides PCR Assay Prgramming 1. Prgram the thermcylcer accrding t the prgram shwn in Table 4 belw. 2. Run PCR. Table 4. C. cladspriides Assay Prgram PCR Cycle Step Temperature Duratin Cycle 1 Step 1 95 C 3 min Cycle 2 (40x) Step 1 94 C 15 sec Step 2 60 C 30 sec Step 3 72 C 45 sec Cycle 3 Step 1 72 C 5 min Cycle 4 Step 1 4 C D. Cladsprium cladspriides PCR Assay Results Interpretatin 1. Fr the analysis f the PCR data, the entire µl PCR Reactin shuld be laded n a 1X TAE 1.7% Agarse DNA gel alng with 10 µl f Nrgen s DNA Marker (prvided). 2. The PCR prducts shuld be reslved n the 1X TAE 1.7% Agarse gel at 150V fr 30 minutes. 3. Sample results are prvided belw: M M Islatin Cntrl (IsC) C.cladspriides Target PCR Cntrl (PCRC) Figure 1: A representative 1X TAE 1.7% agarse gel shwing the amplificatin f Cladsprium cladspriides at different cncentratins (C.cladspriides Target). The size f the C. cladspriides target amplicn crrespnds t 320 bp as represented by the prvided DNA Marker (M). The size f the C. cladspriides Islatin Cntrl (Is C) crrespnds t 550bp as represented by the prvided DNA Marker (M). The CC 2X PCR Master Mix cntains a C. cladspriides PCR Cntrl (PCRC). The C. cladspriides PCRC cntrls fr PCR inhibitin. The size f the C. cladspriides PCRC crrespnds t 150 bp as represented by the prvided DNA Marker (M). The amplificatin frm each lane is interpreted as shwn belw.

7 Lane 1 t 3: Cladsprium cladspriides detected: Lanes shwed the detectin f all three PCR amplicns. Lane 4: N IsC and Template Only C. cladspriides PCRC was detected. Lane 5: C.cladspriides nt detected: Detectin f C. cladspriides IsC and C. cladspriides PCRC, suggesting that the DNA islatin was successful but n C. cladspriides DNA was present in the sample Lane 6: Psitive: All three PCR amplicns were detected Table 5. Interpretatin f PCR Assay Results Input Type C. cladspriides Is C Band (550 bp) C. cladspriides Target Band (320 bp) C. cladspriides PCR C Band (171 bp) Interpretatin Psitive Cntrl X X X Valid Negative Cntrl X X Valid Sample X X X Psitive Sample X X Negative Sample X Negative Sample X X Psitive Sample X X Psitive Sample X Psitive ** Fr results btained that are nt cvered in Table 5 abve, please refer t the Trubleshting Sectin. E. Cladsprium cladspriides PCR Assay Specificity and Sensitivity The specificity f Nrgen s Cladsprium cladspriides PCR Detectin Kit is first and fremst ensured by the selectin f the C.cladspriides -specific primers, as well as the selectin f stringent reactin cnditins. The primers were checked fr pssible hmlgies t all in GenBank published sequences by sequence cmparisn analysis. The specific delectability f all relevant strains has thus been ensured by a database alignment and by PCR amplificatin with the fllwing bacteria cmmnly fund in filed samples. Aspergillus niger Cladsprium sp. Btrytis cinerea Mucr racemsus Alterneria tenuissima Rhizpus ryzae Penicillum sp. Fusarium xysprum

8 F. Linear Range The linear range (analytical measurement) f Nrgen s Cladsprium cladspriides PCR Detectin Kit was determined by analysing a dilutin series f a C. cladspriides quantificatin standards ranging frm 1 x 10 6 cfu/µl t 1 x 10 1 cfu/µl. Each dilutin has been tested in replicates (n = 4) using Nrgen s Cladsprium cladspriides PCR Detectin Kit n a 1X TAE 1.7% agarse gel. The linear range f Nrgen s Cladsprium cladspriides PCR Detectin Kit has been determined t cver cncentratins frm 7 pg t 70 ng Under the cnditins f the Nrgen s C.cladspriides DNA Islatin prcedure, Nrgen s Cladsprium cladspriides PCR Detectin Kit cvers a linear range frm 1500 cpies t 1.5 x 10 6 cpies. Frequently Asked Questins 1. Hw many samples shuld be included per PCR run? Nrgen s Cladsprium cladspriides PCR Detectin Kit is designed t test 24 samples. Fr every 6 samples, a nn-template cntrl (Nuclease Free Water) and a Psitive Cntrl must be included. It is preferable t pl and test 6 samples at a time. If nt, the prvided Psitive Cntrl is enugh t run 3 samples at a time. 2. Hw can I interpret my results if neither the C.cladspriides PCR cntrl nr the C.cladspriides Islatin Cntrl (IsC) amplifies? If neither the C.cladspriides PCR cntrl nr the C.cladspriides Islatin Cntrl (IsC) amplifies, the sample must be re-tested. If the psitive cntrl shwed amplificatin, then the prblem ccurred during the islatin, where as if the Psitive cntrl did nt amplify, therefre the prblem has ccurred during the setup f the PCR assay reactin. 3. Hw shuld it be interpreted if nly the C.cladspriides PCR cntrl shwed amplificatin but neither the C.cladspriides target nr the C.cladspriides Islatin Cntrl (IsC) amplified fr a sample? This indicates a pr islatin. The islatin prcedure must be repeated. 4. Hw shuld it be interpreted if nly the C.cladspriides Islatin Cntrl (IsC) was amplified in a sample? The sample tested can be cnsidered as C.cladspriides negative. 5. Hw shuld it be interpreted if the C.cladspriides PCR cntrl and the C.cladspriides target shwed amplificatin in a sample? The sample tested can be cnsidered psitive. It culd happen when t much template was added t the reactin. 6. Hw shuld it be interpreted if nly the C.cladspriides target and the C.cladspriides PCR cntrl were amplified in a sample? The sample tested can be cnsidered as C.cladspriides psitive. 7. Hw shuld it be interpreted if nly the C.cladspriides target was amplified in a sample? The sample tested shuld be cnsidered as C.cladspriides psitive. At high C.cladspriides cell input, the C.cladspriides amplicn will be predminant and thus the C.cladspriides PCR cntrl as well as the C.cladspriides Islatin cntrl may nt amplify as they cmpete fr PCR resurces. 8. Hw shuld it be interpreted if nly the C.cladspriides PCR cntrl and the C.cladspriides Islatin Cntrl (IsC) shwed amplificatin in a sample? The sample tested can be cnsidered negative

9 9. What if I frgt t d a dry spin after my secnd wash? Yur first DNA elutin will be cntaminated with the Wash Slutin. This may dilute the DNA yield in yur first elutin and it may interfere with the PCR detectin, as ethanl is knwn t be a PCR inhibitr. 10. What if I frgt t add the C.cladspriides Islatin Cntrl (IsC) during the Islatin? It is recmmended that the islatin is repeated. Related Prducts Prduct # Fungi/Yeast Genmic DNA Islatin kit Bacterial Genmic DNA Islatin Kit Plant/Fungi DNA Islatin Kit Technical Assistance NORGEN s Technical Service Department is staffed by experienced scientists with extensive practical and theretical expertise in sample and assay technlgies and the use f NORGEN prducts. If yu have any questins r experience any difficulties regarding Nrgen s Urine DNA Islatin Mini Kit (Slurry Frmat) r NORGEN prducts in general, please d nt hesitate t cntact us. NORGEN custmers are a valuable surce f infrmatin regarding advanced r specialized uses f ur prducts. This infrmatin is helpful t ther scientists as well as t the researchers at NORGEN. We therefre encurage yu t cntact us if yu have any suggestins abut prduct perfrmance r new applicatins and techniques. Fr technical assistance and mre infrmatin, please cntact ur Technical Supprt Team between the hurs f 8:30 and 5:30 (Eastern Standard Time) at (905) r Tll Free at r call ne f the NORGEN lcal distributrs ( r thrugh at techsupprt@nrgenbitek.cm Schmn Parkway, Thrld, ON Canada L2V 4Y6 Phne: (905) Fax: (905) Tll Free in Nrth America: Nrgen Bitek Crp. PI